ALK (Anaplastic Lymphoma Kinase; CD246) is a member of the receptor tyrosine kinase (RTK) family. As a typical member of this family, it is a type-I transmembrane protein essentially consisting of three domains: the extracellular ligand-binding domain (aa19-1038), which contains one LDL-receptor class A domain and two MAM domains (MAM: Meprin, A5 antigen, protein tyrosine phosphatase μ), a transmembrane domain (aa1039-1059) and a cytoplasmic domain (aa 1060-1620), containing the tyrosine kinase domain. A signal peptide is present at the N-terminus of the nascent protein (aa 1-18), which is cleaved upon secretion.
The full-length human and mouse ALK were cloned in 1997 by two independent groups (Iwahara 1997; Morris 1997). ALK is highly similar to the RTK called Leukocyte Tyrosine Kinase (LTK) and belongs to the insulin receptor superfamily. ALK exhibits 57% aa identity and 71% aa similarity with LTK in their regions of overlap (Morris 2001). ALK is highly N-glycosylated and contains 21 putative N-glycosylation sites. Amino acids 687 to 1034 have significant similarity (50% aa identity) to LTK. However, the N-terminus proximal 686 aa sequence shows no homology to any known proteins with the exception of a very short sequence also found in the LDL receptor (Duyster 2001/SWISSPROT). In addition, it contains two MAM domains at aa264-427 and aa478-636 (Meprin, A5 antigen, protein tyrosine phosphatase μ). These domains are thought to have an adhesive function, as they are widespread among various adhesive proteins implicated in cell-to-cell interaction (De Juan 2002). Furthermore, there is a binding site for the ALK putative ligands corresponding to amino acids 396-406 (Stoica 2001; see below). The amino acid sequence of the kinase domain of murine ALK shows 98% aa-identity to human ALK, 78% identity to mouse LTK, 52% to mouse ros, 47% to human insulin-like growth factor receptor and 46% to human insulin receptor (Iwahara 1997; Ladanyi 2000). No splice variants of ALK have been described to date. However, ALK is often associated with chromosomal translocations (see below).
The ALK gene spans about 315 kb and has 26 exons. Much of the gene consists of two large introns that span about 170 kb. The ALK transcript is 6.5 kb of length (Kutok 2002). According to Morris, the cDNA spans 6226 bp (Morris 2001).
ALK expression in mice starts during embryogenesis around the development stage E11 and is persisting in the neonatal periods of development where it is expressed in the nervous system. In the adult, its physiological expression is restricted to certain neuronal (neural and glial cells and probably endothelial cells) regions of the CNS at low levels (Morris 1997; Duyster 2001; Stoica 2001). Actually, the abundance of ALK decreases in the postnatal period (Morris 2001). Based on its expression pattern, a role for the receptor in brain development is suggested (Duyster 2001). The neural-restricted expression of ALK suggests that it serves as a receptor for neurotrophic factors (see later). Consistent with this, its expression pattern overlaps with the genes encoding the TRK family of neurotrophin receptors (Morris 2001). However, ALK knockout mice do not show any obvious phenotype (unpublished data), which might be due to some functional redundancy with TRK family members or other neurotrophin receptors. Notably, hematopoietic tissues show no detectable expression of ALK (see Morris 2001).
Two potential ligands for ALK have recently been described, “pleiotrophin” (PTN) and “midkine” (MK) (Stoica 2001; Duyster 2001; Stoica 2002). The PTN-ALK interaction was identified by using purified human pleiotrophin protein to screen a phage display peptide library. By this method, a sequence of ALK present in its extracellular domain (aa 396-406) was identified. Importantly, this sequence is not shared with LTK, the RTK most closely related to ALK. This ligand-binding region is also conserved in the potential homologue of ALK in Drosophila (Loren 2001). ALK is phosphorylated rapidly upon PTN binding (Bowden 2002). Moreover, ALK has been shown to be stimulated by pleiotrophin in cell culture. This makes the pleiotrophin/ALK interaction particularly interesting in the light of the pathological implications pleiotrophin has (Stoica 2001). Cell lines that lack ALK expression also fail to show a growth response to pleiotrophin and vice versa (Stoica 2001). In vivo, elevated pleiotrophin levels in the serum of patients suffering from various solid tumors have been demonstrated, and animal studies have suggested a contribution of pleiotrophin to tumor growth (Stoica 2001). The role of PTN as rate-limiting angiogenic factor in tumor growth is well established in animal models (Choudhuri 1997). In 1996 Czubayko et al. demonstrated the importance of PTN in tumor angiogenesis, in prevention of apoptosis and metastasis by modulating PTN levels with a ribozyme targeting approach (Czubayko 1996). Serum level measurements of PTN in mice demonstrated a clear correlation with the size of the tumor. PTN plays a significant role in some of the most aggressive human cancer types such as melanoma and pancreatic cancer thus giving interesting perspectives for potential further applications of an ALK inhibitor (Weber 2000; Stoica 2001). In human patients, elevated serum pleiotrophin levels were found in patients with pancreatic cancer (n=41; P<0.0001) and colon cancer (n=65; P=0.0079). In healthy individuals, PTN is expressed in a tightly regulated manner during perinatal organ development and in selective populations of neurons and glia in the adult.
Co-expression of PTN and ALK, as found in several cancer cell lines, indicates that they could form an autocrine loop of growth stimulation (Stoica 2001). In spite of all these data, the literature indicates that is not yet clear if the effects of PTN are mediated by ALK alone and/or by other unidentified PTN receptors (Duyster 2001). At least two other potential receptors of PTN have been suggested: the receptor tyrosine phosphatase RPTPβ and the heparan sulfate proteoglycan N-syndecan. However, RPTPβ might act as a signalling modulator of PTN/ALK signalling and N-syndecan as a chaperone for the ligand (Bowden 2002).
Recently, another secreted growth factor related to pleiotrophin called midkine (MK) has been identified as a second ligand for ALK. Similarly to PTN, binding and activating functions (e.g. induction of soft agar colony formation in cell cultures) of MK can be blocked by the same antibody raised against the ALK-ECD (Stoica 2001). Like pleiotrophin, midkine is upregulated in many tumors, although its physiological expression is very restricted in adult normal tissues (Stoica 2002). Analysis of 47 bladder tumor samples revealed that MK expression is significantly (about four times) enhanced as compared to normal bladder tissue. Furthermore, pronounced overexpression correlates with poor patient survival (O'Brien 1996).
However, the affinity of MK for ALK is about 5 times lower than the one of pleiotrophin (Stoica 2002). Interestingly, as with pleiotrophin, inhibition of ALK via ribozymes also inhibits the effects of MK in cell culture (Stoica 2002). The authors of these studies also come to the conclusion that inhibition of the PTK/MK/ALK pathway opens very attractive possibilities for the treatment of various diseases, some of them having very limited treatment options so far, such as, for example, glioblastoma and pancreatic cancer. (Stoica 2002).
In healthy individuals, ALK mRNA expression peaks during the neonatal period and persists in adults in a few selected portions of the nervous system. Recently, expression of the ALK protein was also detected in endothelial cells that were associated to neuronal and glial cells. Evidence that at least a part of the malignant activities described for pleiotrophin are mediated through ALK came from experiments in which the expression of ALK was depleted by a ribozyme targeting approach. Such depletion of ALK prevented pleiotrophin-stimulated phosphorylation of the anti-apoptopic protein Akt and led to a prolonged survival of mice that had received xenografts. Indeed, the number of apoptopic cells in the tumor grafts was significantly increased, when ALK expression was depleted (Powers 2002).
Evidence that malignant activities described for MK are mediated through ALK came from experiments with monoclonal antibodies directed against the ALK ECD. Addition of a 1:25 dilution of hybridoma cell supernatant from two anti-ALK ECD antibodies leads to a significant decrease in colony formation of SW-13 cells in soft agar (Stoica 2002). Analysis of ten different cell lines revealed that the ability for a growth response to PTN perfectly correlated with the expression of ALK mRNA (the following cell lines responded to PTN and were found to express ALK mRNA: HUVEC, NIH3T3, SW-13, Colo357, ME-180, U87, MD-MB 231; Stoica 2001). Interestingly, in some cancer cell lines (Colo357 pancreatic cancer, Hs578T breast cancer and U87 glioblastoma), PTN and ALK are co-expressed, indicating that PTN and ALK form an autocrine loop of growth stimulation (Stoica 2001).
Interestingly, both PTN and MK have been shown to cause transcriptional up-regulation of the anti-apoptotic bcl-2 protein (Stoica 2002). In addition activated Akt (which is a crucial downstream target of aberrant ALK signalling) phosphorylates the pro-apoptotic factor called bad, thus leading to dissociation from bcl-xl, which, when liberated from bad, can suppress apoptosis by blocking the release of cytochrome c (see Bowden 2002 for references).
Aberrant expression of ALK might be involved in the development of several cancers. However, it was first associated with a subgroup of high-malignant Non-Hodgkin lymphomas (NHLs), the so-called Anaplastic Large Cell Lymphomas (ALCLs). Non-Hodgkin lymphomas represent clonal neoplasias originating from various cells of lymphatic origin.
Most patients with the primary systemic clinical subtype of ALCL have the t2,5 translocation, expressing a fusion protein that joins the N-terminus of nucleophosmin (NPM) to the C-terminus of ALK. The fusion consists of aa 1-117 of NPM fused to aa 1058-1620 of ALK and the chromosomal breakage is located in an intron located between the exons encoding the TM and juxtamembrane domain of ALK (Duyster 2001). NPM-ALK is a transcript containing an ORF of 2040 bp encoding a 680aa protein (Morris 2001). This corresponds to a breakage in intron 4 of NPM, which spans 911 bp and intron 16 of ALK which spans 2094 bp (Kutok 2002). Most likely the ALK sequence in this fusion protein is the minimal sequence required for the protein to lead to ALCL (Duyster 2001). The inverse fusion (ALK-NPM) is not expressed, at least not in lymphoid cells (Kutok 2002). The wild-type NPM protein demonstrates ubiquitous expression and functions as a carrier of proteins from the cytoplasm into the nucleolus. As a matter of fact, NPM is a 38 kDa nuclear protein encoded on chromosome 5 that contains a NLS, binds nuclear proteins and engages in cytoplasm/nuclear trafficking (Duyster 2001). NPM is one of the most abundant nucleolar proteins and is normally present as a hexamer (Morris 2001). Most importantly NPM normally undergoes self-oligomerization (hexamers) as well as hetero-oligomerization with NPM-ALK (Duyster 2001). The 2;5 translocation brings the ALK gene portion encoding the tyrosine kinase on chromosome 2 under the control of the strong NPM promoter on chromosome 5, producing permanent expression of the chimeric NPM-ALK protein (p80) (Duyster 2001). Hence, ALK kinase is deregulated and ectopic, both in terms of cell type (lymphoid) and cellular compartment (nucleus/nucleolus and cytoplasm) (Ladanyi 2000). The localization (cytoplasm or nucleus) of NPM seems not to affect its effect on lymphomagenesis (Duyster 2001). The resultant aberrant tyrosine kinase activity triggers malignant transformation via constitutive phosphorylation of intracellular targets. Various other less common ALK fusion proteins are associated with ALCL. All variants demonstrate linkage of the ALK tyrosine kinase domain to an alternative promoter that regulates its expression.
Full-length ALK has been reported to be also expressed in about 92% of primary neuroblastoma cells and in some rhabdomyosarcomas (Lamant 2000). However, no correlation between ALK expression and tumor biology has been demonstrated so far. This fact, taken together with the lack of evidence regarding significant levels of endogenously phosphorylated ALK in these tumors, suggest that ALK expression in neuroblastoma reflects its normal expression in immature neural cells rather than a primary oncogenic role and ALK in these tumors is not constitutively phosphorylated thus questioning an important role for ALK in these tumors (Duyster 2001; Pulford 2001). Nevertheless, ALK signalling might be important in at least some neuroblastomas, as suggested by Miyake et al., who found overexpression and constitutive phosphorylation of ALK due to gene amplification in neuroblastoma-derived cell lines (Miyake 2002). However, other neuroblastoma-derived cell lines do not show constitutive activation of ALK, thus arguing against a general pathological involvement of ALK (Dirks 2002; Pulford 2004).
Most interestingly, ALK seems to be important for growth of glioblastoma multiforme, a highly malignant brain tumor that offers very limited therapeutic options (Powers 2002). Multiple genetic alterations have been shown to occur in these devastating tumors including loss or mutations of PTEN, p53 and INK4a-ARF. In addition, RTK signalling plays a particularly important role in growth and development of these tumors, which overexpress various growth factors such as PDGF, HGF, NGF and VEGF suggesting autocrine RTK signalling loops. Powers and colleagues have shown mRNA and protein expression of ALK in glioblastoma patient tumor samples, whereas the signals were not detectable in normal adjacent brain tissue (Powers 2002). Furthermore, human U87MG glioblastoma cells (which are derived from a patient and represent a well-characterized model system to study tumorigenesis and signalling in glioblastoma) show ALK-dependent anti-apoptotic behaviour in xenograft studies. When ALK is depleted in these tumor cells by the use of ribozymes, mice injected with these tumor cells survive at least twice as long as when injected with wild-type tumor cells, and these tumor cells show drastically increased apoptosis. Thus, ALK and its ligand(s) provide an essential survival signal that is rate-limiting for tumor growth of U87MG cells in vivo (Powers 2002). These finding indicate that inhibition of ALK signalling could be a promising approach to improve life expectancy of glioblastoma patients.
Glioblastoma multiforme is by far the most common and malignant primary glial tumor with an incidence of about 2/100'000/y (about 15'000 cases in US and Western Europe per year). It affects preferentially the cerebral hemispheres, but can also affect the brain stem (mainly in children) or the spinal cord. The tumors may manifest de novo (primary glioblastoma) or may develop from lower grade astrocytomas (secondary glioblastoma). Primary and secondary glioblastomas show little molecular overlap and constitute different disease entities on molecular level. They both contain many genetic abnormalities including affection of p53, EGFR, MDM2, PDGF, PTEN, p16, RB.
No significant therapy advancement has occurred in the last 25 years. Therapies are only palliative and can expand the life expectance from 3 months to 1 year. Patients usually present with slowly progressive neurological deficit, e.g. motor weakness, intracranial pressure symptoms, e.g. headache, nausea, vomiting, cognitive impairment, or seizures. Changes in personality can also be early signs. The etiology of glioblastoma is unknown, familial cases represent less than 1%. The only consistent risk factor identified is exposure to petrochemicals. Diagnosis is made mainly by imaging studies (CT, NMR) and biopsy. Completely staging most glioblastomas is neither practical nor possible because these tumors do not have clearly defined margins. Rather they exhibit well-known tendencies to invade locally and spread along compact white matter pathways. The primary reason why no curative treatment is possible is because the tumor is beyond the reach of local control when diagnosed. The primary chemotherapeutic agents are carmustine (an alkylating agent) and cisplatinum but only 40% of patients show some response.
Although there are quite some uncertainties regarding the role of ALK in glioblastoma, this disease offers various approaches for ALK-directed drugs. In fact, for this devastating disease even a small improvement of current therapy options would serve an enormous medical need. It is important to note that since glioblastoma cells express the full-length ALK, for treating this cancer ALK could be considered as a target not only for small molecule kinase inhibitors but also for antibodies and/or antibody fragments such as scFvs i.e. to induce apoptosis of tumor cells. The strict localization of glioblastoma to the CNS supports the use of scFvs, if they can be delivered efficiently to the CNS (no rapid clearance due to compartmentalization, but better tumor penetration compared to IgGs due to their smaller size). Antibodies and/or antibody fragments could be directed against the ligand-binding sequence of ALK (aa 396-406) or against other parts of the extracellular parts of the receptor.
The very limited expression of ALK in healthy tissues under physiological conditions indicates that tumors expressing ALK might be an excellent target for disease treatment using radioactive or toxin-labelled antibodies and/or antibody fragments, irrespective of whether ALK is involved in the pathogenesis of these tumors or not. In addition to glioblastoma cells, ALK expression has been found with high significance in melanoma cell lines and breast carcinoma cell lines (without being constitutively phosphorylated) (Dirks 2002). The fact that a large portion of the extracellular domain of ALK seems to be rather unique in the human proteome should make this approach highly specific.
WO9515331/U.S. Pat. No. 5,529,925 discloses the cloning and sequencing of the human nucleic acid sequences, which are rearranged in the t(2; 5)(p23; q35) chromosomal translocation event which occurs in human t(2; 5) lymphoma. The rearrangement was found to bring sequences from the nucleolar phosphoprotein gene (the NPM gene) on chromosome 5q35 to those from a previously unidentified protein tyrosine kinase gene (hereinafter the ALK gene) on chromosome 2p23. The sequence of the fusion gene and fusion protein (NPM/ALK fusion gene or protein, respectively) were also disclosed.
The full-length ALK sequence is patented in U.S. Pat. No. 5,770,421, entitled “Human ALK Protein Tyrosine Kinase.” Furthermore, the U.S. Pat. No. 6,174,674B1 entitled “Method of detecting a chromosomal rearrangement involving a breakpoint in the ALK or NPM gene”, discloses primers for detecting the NPM-ALK fusion sequence in patient samples. In another patent, U.S. Pat. No. 6,696,548 entitled “ALK protein tyrosine kinase/receptor and ligands thereof”, the use of ALK for detection of ALK ligands and antibodies binding to specific sequences of ALK is disclosed. It also discloses a method of identifying an agent capable of binding to the isolated ALK polypeptide. WO0196394/US20020034768 discloses ALK as receptor of pleiotrophin. US20040234519 discloses anti-pleiotrophin antibodies, and WO2006020684 describes the detection of pleitrophin.